Kenneth A. Alexander

RNA Interference of Human Papillomavirus Type 18 E6 and E7 Induces Senescence in HeLa Cells

ABSTRACT The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.

Infections diagnosed in the first year after pediatric stem cell transplantation

Background. Cumulative incidence of infections in the first year posttransplantation in adult patients has been well-described. Such description is less than complete for pediatric stem cell transplantation (SCT) patients. Further among those patients who have been infected, analysis of risk factors for infection has not been well-described for a large cohort of pediatric SCT patients. Methods. We conducted a retrospective cohort study of infections in the first year after SCT at Duke University Medical Center. We recorded all infections in the first year after transplantation. We determined incidences for 6 categories of infection: Gram-negative rods; Gram-positive cocci; yeast species;Aspergillus sp.; adenovirus; and cytomegalovirus. We determined incidences based on type of transplant and days post transplantation. We also completed bivariable and multivariable analysis of risk factors [neutropenia, graft vs. host disease (GVHD) and GVHD treatment] for infection type among those children who were infected. Results. We evaluated 510 transplants in 485 children. There were 584 infections in the first year after transplantation. During the first 30 days posttransplantation, type of transplantation did not predict incidence of infection or type of infection. After 30 days children who received unrelated cord blood transplant and matched unrelated donor transplant were at much higher risk of infection than were patients who received autologous, matched sibling or haploidentical transplant (P < 0.001). Patients who received unrelated cord blood or matched unrelated donor transplantation were at higher risk of aspergillosis (P = 0.002), candidiasis (P = 0.005) and adenovirus (P < 0.0001) but not cytomegalovirus (P = 0.18). In analysis of risk factors among those infected, patients with aspergillosis were more likely to have severe GVHD: multivariable 1 year risk ratio, 7.5; 95% confidence interval, 3.0,18.4. Neutropenia was more strongly associated with Gram-negative rod infection than any other type of infection. Conclusions. The incidence of infection immediately after transplantation did not differ significantly by type of transplant in this pediatric population. Type of transplant predicted increased incidence of infection 30 days posttransplantation and increased incidence of infection with several organisms traditionally associated with a high mortality rate in the transplant population.

Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction

Using yeast forward and reverse two-hybrid analyses, we have discovered that the replication terminator protein Tus of Escherichia coli physically interacts with DnaB helicase in vivo. We have confirmed this protein–protein interaction in vitro. We show further that replication termination involves protein–protein interaction between Tus and DnaB at a critical region of Tus protein, called the L1 loop. Several mutations located in the L1 loop region not only reduced the protein–protein interaction but also eliminated or reduced the ability of the mutant forms of Tus to arrest DnaB at a Ter site. At least one mutation, E49K, significantly reduced Tus–DnaB interaction and almost completely eliminated the contrahelicase activity of Tus protein in vitro without significantly reducing the affinity of the mutant form of Tus for Ter DNA, in comparison with the wild-type protein. The results, considered along with the crystal structure of Tus–Ter complex, not only elucidate further the mechanism of helicase arrest but also explain the molecular basis of polarity of replication fork arrest at Ter sites.

Antiviral Therapy for Human Papillomaviruses: Rationale and Prospects

It is not generally appreciated that the human papillomaviruses (HPVs) are the most common sexually transmitted viral agents in the United States, infecting as many as 5% to 20% of persons aged 15 to 49 years [1]. Because genital HPV infection is not a reportable disease, accurate epidemiologic data are not readily available; however, the Centers for Disease Control and Prevention has estimated that nearly 1 million new cases of genital warts are diagnosed every year in the United States alone [2]. As with other sexually transmitted diseases, HPV infection often occurs in association with diseases such as syphilis, gonorrhea, chlamydia, herpes simplex virus (HSV), and human immunodeficiency virus (HIV). Human papillomavirus infection is common across all races and socioeconomic groups and is prevalent throughout the world in sexually active persons. In the last 30 years, the incidence of genital HPV infection has increased dramatically. It has been estimated that the number of physician visits in the United States for genital warts increased from fewer than 200 000 in 1966 to 1.15 million in 1984 [3, 4]. This incidence has continued to increase throughout the latter part of the 1980s, with almost 1.5 million consultations annually [5]. The dramatic increase in the number of patients seeking diagnosis and therapy for HPV infection may be partly due to an increased awareness and recognition by both the public and clinicians. The highest prevalence of genital HPV infection is found in sexually active women younger than age 25 years. Polymerase chain reaction analysis of genital specimens from women attending university infirmaries indicated that as many as 40% had some evidence of HPV infection [6]. A hybridization analysis for viral DNA found that 15% of adolescent females attending family planning clinics had evidence of an HPV infection and that 76% of the HPV-positive females had a normal Papanicolaou smear result [7]. A separate comparative analysis of 454 women attending a sexually transmitted diseases clinic and 545 college women showed that 11% of the patients with a sexually transmitted disease but only 2% of the college students presented with visible genital warts. In this study, however, analysis of cervical specimens from women without visible lesions showed that 10% of the patients with a sexually transmitted disease and 11% of the students were positive for HPV DNA or antigen [8]. Although the prevalence of asymptomatic or clinically inapparent disease is uncertain, clearly a substantial proportion of infected persons progress to condylomata or to cervicovaginal dysplasia and cervical cancer. The greatest prevalence of high-grade cervical dysplasia is found in women older than age 25 years, with the peak incidence of cervical cancer occurring after age 35 years [9]. Because most cases of cervical dysplasia and carcinoma (approximately 90%) are associated with HPV infection, the increasing prevalence of genital warts in young women may portend a future epidemic increase in the rate of premalignant disease and cervical cancer. Despite the high prevalence of HPV infection, no available drug therapy effectively eliminates HPV infection and replication or prevents HPV-associated malignant progression. As a consequence, there is a striking unmet medical need for the development of safe and effective therapies for HPV-associated disease. Current treatment relies on excision or ablation of dysplastic or malignant tissue and is associated with substantial rates of recurrence, discomfort, and expense [10, 11]. Although papillomaviruses cannot yet be routinely cultured in the laboratory, the tools of molecular virology have been used extensively in the last 10 years to study the functions of the 8 to 10 individual viral genes. Several of the viral gene products represent attractive targets for the development of antiviral inhibitors. We examine the current state of antiviral investigations and the prospects for the development of potent and clinically useful therapies for inhibiting HPV replication and pathology. General Properties of the Human Papillomaviruses The papillomaviruses are members of the Papovaviridae family and infect most vertebrate species with exclusive host and tissue specificity. The virion is a small, nonenveloped icosahedral capsid that contains a single molecule of supercoiled, double-stranded, circular DNA about 8000 base-pairs in length. Viral DNA is replicated in the nuclei of infected cells in association with low-molecular-weight histones and is organized into nucleosomal structures that are reminiscent of host cellular chromatin. The genomes of many of the animal and human papillomaviruses have been completely sequenced. The arrangement of the open-reading frames, which are translated into viral proteins, and the nucleotide and amino-acid sequences of certain domains of the viral proteins have been well conserved during evolution of the animal and human viruses. As shown in Figure 1, the viral genome can be bisected into early (E1 to E7) and late regions (L1 and L2) separated by a 1-kb long control region (LCR), which contains transcriptional control elements and the origin of viral DNA replication. By convention established for other DNA viruses, viral genes designated as early encode functions that are important for the establishment of infection and for the initiation of viral DNA replication. The late genes encode the viral capsid proteins, the expression of which is normally delayed until after viral DNA is amplified [12]. Figure 1. A circular map of the double-stranded genome of human papillomavirus type 11 (HPV-11) (229). For many years, it was assumed that all infectious human epidermal lesions were caused by a single human wart virus. It was not until the mid-1970s that restriction-endonuclease mapping and hybridization analysis provided the earliest indication of the remarkable plurality of HPV genotypes. Approximately 70 different HPV types are currently recognized and have been isolated from various mucosal and cutaneous epithelial lesions (Table 1). Human papillomaviruses are normally grouped according to their pathologic associations and tissue specificity as cutaneous or mucosal [13]. The 23 mucosal-associated HPVs can be further subdivided into those at high risk for malignant progression and those at low risk for progression [14]. Table 1. Human Papillomaviruses Associated with Human Disease Nonmalignant Human Papillomavirus Disease Cutaneous Warts The most common and familiar result of HPV infection is the development of a localized, benign epithelial neoplasm or wart. Cutaneous palmar and plantar warts are widespread in the population and are typically found to contain HPV types 1, 2, 3, 4, or 10. Cutaneous warts can persist or grow slowly for years and then abruptly disappear without apparent cause or therapy. The incidence of benign warts is highest in children and young adults and decreases later in life, presumably because of the development of systemic immunity. Plantar and palmar warts are considered to be exclusively benign. Considering the ubiquity of common skin warts in humans and the relation of other types of HPV to cancer, it is remarkable that the reported incidence of malignant conversion of skin warts is essentially 0. In addition, the sites of infection, commonly the hands and feet, are frequently exposed to mechanical trauma or other potential cocarcinogenic factorshumans are often in contact with the environment through bare hands and feet. These observations show and support a seminal theme in the pathobiology of HPV disease: The potential for malignant conversion of HPV-associated lesions is, at least in part, determined by the infecting HPV genotype and its target tissue. Condyloma Acuminata In the United States, the incidence of HPV-associated genital warts is increasing dramatically. Ranking only behind chlamydial infection and gonorrhea, sexually transmitted genital warts are one of the fastest growing sexually transmitted diseases in the United States, with an estimated incidence two to three times that of genital herpes. Clusters of exophytic condylomata generally develop on the penis, vagina, vulva, and perineum and around the anus. Only rarely do condylomata develop on the cervix. More than 90% of the lesions examined are associated with infection by the low-risk HPV types 6 and 11, the same two virus types associated with respiratory papillomatosis [15-17]. Condylomata may spontaneously regress or persist for years. Progression to invasive carcinoma is seen at a relatively low frequency [14]. During pregnancy, condylomata have been observed to increase in both size and number, possibly because of the stimulatory effect of steroid hormones on HPV transcription [18, 19] or because of a decrease in host-cell-mediated immunity. Benign condylomata are often resistant to various forms of therapy, which results in high recurrence rates (30% to 90%). In addition to the social stigma associated with them, genital warts can be painful, may spread locally in an individual patient, and are infectious to sexual partners. Approximately two thirds of the sexual partners of patients with condylomata have been observed to develop genital warts during a 1- to 8-month observation period [5]. Although condylomata are not generally considered life-threatening, without effective antiviral therapy they represent a substantial problem in therapeutic and epidemiologic management. Laryngeal Papillomas Laryngeal or recurrent respiratory papillomas are benign epithelial tumors that usually develop on the larynx and are associated with infection by HPV types 6 and 11 [20, 21]. Although spontaneous malignant conversion is rare [22], therapeutic irradiation of juvenile laryngeal papillomas has been associated with a high incidence of carcinoma 5 to 40 years after treatment [23]. More recently, HPVs have been identified in lesions of the nose and oral cavity and h

Human TATA Binding Protein Inhibits Human Papillomavirus Type 11 DNA Replication by Antagonizing E1-E2 Protein Complex Formation on the Viral Origin of Replication

ABSTRACT The human papillomavirus (HPV) protein E2 possesses dual roles in the viral life cycle. By interacting directly with host transcription factors in basal keratinocytes, E2 promotes viral transcription. As keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, to activate vegetative viral DNA replication. How E2s major role switches from transcription to replication during keratinocyte differentiation is not understood, but the presence of a TATA site near the viral origin of replication led us to hypothesize that TATA-binding protein (TBP) could affect HPV replication. Here we show that the C-terminal domain of TBP (TBPc) is a potent inhibitor of E2-stimulated HPV DNA replication in vitro (50% inhibitory concentration = 0.56 nM). Increasing the E1 concentration could not overcome TBPc inhibition in replication assays, indicating that TBPc is a noncompetitive inhibitor of E1 binding. While direct E2-TBPc association could be demonstrated, this interaction could not fully account for the mechanism of TBPc-mediated inhibition of viral replication. Because E2 supports sequence-specific binding of E1 to the viral ori, we proposed that TBPc antagonizes E1-ori association indirectly through inhibition of E2-DNA binding. Indeed, TBPc potently antagonized E2 binding to DNA in the absence (Ki = 0.5 ± 0.1 nM) and presence (Ki = 0.6 ± 0.3 nM) of E1. Since E2 and TBPc cannot be coadjacent on viral sequences, direct DNA-binding competition between TBPc and E2 was responsible for replication inhibition. Given the ability of TBPc to inhibit HPV DNA replication in vitro and data indicating that TBPc antagonized E2-ori association, we propose that transcription factors regulate HPV DNA replication as well as viral transcription.

A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase

The current models that have been proposed to explain the mechanism of replication termination are (i) passive arrest of a replication fork by the terminus (Ter) DNA-terminator protein complex that impedes the replication fork and the replicative helicase in a polar fashion and (ii) an active barrier model in which the Ter-terminator protein complex arrests a fork not only by DNA-protein interaction but also by mechanistically significant terminator protein-helicase interaction. Despite the existence of some evidence supporting in vitro interaction between the replication terminator protein (RTP) and DnaB helicase, there has been continuing debate in the literature questioning the validity of the protein-protein interaction model. The objective of the present work was two-fold: (i) to reexamine the question of RTP-DnaB interaction by additional techniques and different mutant forms of RTP, and (ii) to investigate if a common domain of RTP is involved in the arrest of both helicase and RNA polymerase. The results validate and confirm the RTP-DnaB interaction in vitro and suggest a critical role for this interaction in replication fork arrest. The results also show that the Tyr33 residue of RTP plays a critical role both in the arrest of helicase and RNA polymerase.

Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein.

ABSTRACT Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

Recent advances in diagnosis and therapy of human papillomaviruses

Infection with human papillomavirus is extremely common throughout the world. Almost 50% of sexually active young women are infected with human papillomavirus and although most infections are transient, a subset has the potential to progress to invasive cancer. During the last 20 years, our understanding of the human papillomavirus life cycle and the role of human papillomavirus in human cancer has dramatically increased. Recent technological advances in human papillomavirus detection have provided the means to detect the presence of human papillomavirus with great sensitivity. In the context of patient care, there is still substantial debate regarding the optimal diagnostic and prognostic use of information derived from hybrid capture or polymerase chain reaction-based detection. The inventory of available treatment options is growing somewhat slowly. The most promising advances are being made in the clinical evaluation of candidates for prophylactic vaccination. This review is focused on the current status and future directions of prevention, diagnosis and therapy.